Chemical synthesized peptides were expected to be the third milestone, following inactivated virus vaccines and virus subunit vaccines, in the history of human vaccine development. However, none of preventative or therapeutic synthetic peptide vaccines comprising one or two epitopes (epitope is the part of an antigen that is recognized by the immune system, specifically by antibodies and B cells. See the definition in the DETAILED DESCRIPTION OF THE INVENTION) have been approved for clinical applications since 1970s due to the lack of sufficient and efficient immune effects against viruses. Therefore, in order to achieve sufficient and efficient preventive effects against viruses for clinical applications, synthetic peptide vaccines should combine more linear B-cell epitopes if possible (multi-epitope peptide immunogen can be prepared with bioengineering). In addition, single-epitope peptides consisting of 12˜20 amino acids are usually used as antigen reagents to detect serum antibodies for the diagnosis of virus infections because natural virus proteins are usually unavailable.
It has been known that a linear B-cell epitope usually consists of 3˜8 amino acids. Therefore, it is obvious that using a longer peptide as testing antigen could have problems because it might contain additional epitopes that are recognized by specific antibodies among thousands of antibodies that are present in normal human serum, resulting in high false positive detection rates. In order to improve the specificity and sensitivity of the epitope peptide antigens in diagnosing virus infections and autoimmune diseases, a new trend has been to use short epitope peptides based on minimal motifs, and/or to develop a multi-epitope peptide antigen comprising as many precise epitope motifs as possible on a specific target antigen protein. The former strategy could avoid false positive results, and the rafter will improve detection sensitivity since multiple epitopes in a target protein would be recognized by more antibodies, and this strategy is particularly useful for detecting lower antibody titers elicited by viral infections or autoimmune diseases.
In addition, using the short epitope peptides based on precise motifs also will improve the effectiveness of making monoclonal or polyclonal antibodies for the development of therapeutic biologics or detection agents. Obviously, whether these goals can be achieved by using short epitope peptides or multi-epitope peptides to develop detection antigen, vaccines or therapeutic biologics, will first depend on the identification of a complete set of linear B-cell epitopes and their precise motifs in a target antigen protein.
A large amount of research data demonstrated that cervical cancer, which ranks 2nd globally in the death rates of female cancers, is 90% related to persisted infection at the epidermal of gynecological tract with high-risk (HR-) HPV subtypes, such as HPV 16, 18, 31 and 45 (1). Currently, two pharmaceutical companies (Merck in the U.S. and GlaxoSmithKline (GSK) in UK] have developed preventative HPV vaccines that comprise L1 proteins of HPV 6, 11, 16 and 18, and L1 protein of HPV 16 and 18-L1 proteins, respectively. Even though much research has been done in developing HPV peptide vaccines, there is no report of market approval regarding these peptide vaccines.
Because most women infected by HPV can usually automatically eliminate the infected HPV, and only those, who are continuously infected by HPV, will resulted in cervical cancer, it becomes very important to develop inexpensive and convenient serum detection for HPV infection. Obviously, screening with such detection kits has great significance because such screening can determine who are the objects of vaccination with specific preventative or therapeutic HPV vaccines and what types of specific HPV vaccines should be used. Because of the limitations of early methodologies for epitope identification, it is almost impossible to identify a complete set of linear epitopes (particularly their minimal motifs) on a target protein. Identification of such epitopes or their minimal motifs can improve the detection accuracy, avoiding cross reactions with one or some of thousands of antibodies against unknown antigens present in normal human serum. Currently, no sensitive specific serological detection method for HPV infection has been established, even though in the early time, HPV-infected serum was used to identify specific markers for serological detection of HPV infections (2-5).
HPV 58 is one of the high-risk (HR)-HPVs after HPV 16 and 18 (6). Recent research also demonstrated that high positive detection rates for HPV 58 have been shown in cervical cancer patients in many areas of China including Jiangxi, Shanghai, Hebei, Shanxi, Beijing, Hong Kong and Taiwan (7-15). In some areas of China the detection rates for HPV 58 is even higher than those for HPV18 and only secondary to HPV16 (16-17).